Alternative pathway of H2S and polysulfides production from sulfurated catalytic-cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase

Article abstract of DOI:10.1016/j.bbrc.2018.01.056

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H₂S_n revealed that H₂S₂ first appeared, and then H₂S and H₂S₃ did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.

Full text of DOI:10.1016/j.bbrc.2018.01.056

Accepted Manuscript
Alternative pathway of H S and polysulfides production from sulfurated catalytic-
2
cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase
Noriyuki Nagahara, Shin Koike, Takashi Nirasawa, Hideo Kimura, Yuki Ogasawara
PII:
S0006-291X(18)30062-7
10.1016/j.bbrc.2018.01.056
YBBRC 39229
DOI:
Reference:
To appear in: Biochemical and Biophysical Research Communications
Received Date: 25 December 2017
Accepted Date: 9 January 2018
Please cite this article as: N. Nagahara, S. Koike, T. Nirasawa, H. Kimura, Y. Ogasawara, Alternative
pathway of H S and polysulfides production from sulfurated catalytic-cysteine of reaction intermediates
2
of 3-mercaptopyruvate sulfurtransferase, Biochemical and Biophysical Research Communications
(2018), doi: 10.1016/j.bbrc.2018.01.056.
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ACCEPTED MANUSCRIPT
Alternative pathway of H2S and polysulfides production from sulfurated
catalytic-cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase
Noriyuki Nagaharaa*, Shin Koikeb, Takashi Nirasawac, Hideo Kimurad, Yuki
Ogasawarab
aIsotope Research Center, Nippon Medical School
bDepartment of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1
Noshio,
Kiyose-shi,
Tokyo
204-8588,
Japan
(skoike@my-pharm.ac.jp)
(yo@my-pharm.ac.jp)
cBruker Daltonics K.K., 3-9 Moriyacho Kanagawa-ku, Yokohama 221-0022, Japan
(Takashi.Nirasawa@bruker.com)
dDepartment of Molecular Pharmacology, National Institute of Neuroscience, National
Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502,
Japan (kimura@ncnp.go.jp)
*Address of corresponding author: Dr. Noriyuki Nagahara,
Isotope Research Center, Nippon Medical School
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1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
Tel.: +81 3 3822 2131; Fax: + 81 3 5685 3065; E-mail: noriyuki @ nms.ac.jp
Short title
Alternative H2Sn production from reaction intermediates of MST
Keywords
Hydrogen sulfide; Monobromobimane; 3-Mercaptopyruvate sulfurtransferase;
Polysulfide; Reaction intermediate; Thiosulfate
Abbreviations
CN, cyanide, ESI, electrospray ionization, FL, fluorescence, HPLC, high-pressure liquid
chromatography, MALDI-TOF/TOF MS, Matrix-assisted laser desorption ionization
tandem time-of-flight mass spectrometry, mBB, monobromobimane, MS/MS, tandem
mass spectrometry, MST, 3-mercaptopyruvate sulfurtransferase, TDR, thioredoxin
reductase, Trx, thioredoxin, TS, thiosulfate
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Abstract
It has been known that hydrogen sulfide and/or polysulfides are produced from a
(poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase
(MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the
donor substrate and the catalytic reaction was terminated on the formation of a persulfide or
polysulfides. We can present alternative pathway of production of hydrogen sulfide
and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction
intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization
tandem time-of-flight mass spectrometric analysis revealed that after prolonged
incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the
sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with
reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH =
1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric
analysis for monobromobimane-derivatized H2Sn revealed that H2S2 first appeared, and
then H2S and H2S3 did later. The results were confirmed by high-performance liquid
chromatography-fluorescence analysis.
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Highlights
1. Thiosulfate forms (poly)sulfurated catalytic-site cysteine of reaction intermediate of
3-mercaptopyruvate sulfurtransferase and the catalytic reaction was terminated on the
formation of a persulfide or polysulfide.
2. (Poly)sulfurated catalytic-site cysteine can be reduced by thioredoxin (Trx) with
reducing system (Trx, Trx reductase, and NADPH).
3. Hydrogen sulfide and/or polysulfides are alternatively produced from a
(poly)sulfurated reaction intermediate.
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1. Introduction
Hydrogen sulfide and polysulfides have been reported to perform important
physiological functions and are produced by cystathionine β-synthase (EC 4.2.1.22) [1],
cystathionine -lyase (CSE, EC 4.4.1.1) [2], 3-mercaptopyruvate sulfurtransferase
(MST, EC 2.8.1.2) [3,4], and thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1) [5].
MST catalyzes a trans-sulfuration reaction from mercaptopyruvate to thiol-containing
compounds. During this catalytic reaction, a persulfide is formed at the catalytic-site
cysteine as a reaction intermediate [6,7]. Then, an acceptor substrate, a thiol-containing
compound, attacks the outer sulfur of the persulfide, and then it is transferred to the
substrate thus forming a new persulfide. Hydrogen sulfide and polysulfides can be
produced during these reactions. Namely, they are produced from polysulfurated
acceptor substrates via direct reduction by thioredoxin (Trx). Mikami’s group
demonstrated that MST produces hydrogen disulfide in vivo [5, 8]. Yadav’ group
demonstrated that human MST did by enzyme kinetics study [9]. Nonetheless, one
possibility is that they are produced from a polysulfurated catalytic-site cysteine via
direct reduction by Trx. In the MST-catalyzed reaction, mercaptopyruvate functions as
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both the donor and acceptor substrate. However, in the present study, we used
thiosulfate (TS) as the donor substrate and the catalytic reaction was terminated on the
formation of a persulfide or polysulfide. We can therefore demonstrate that hydrogen
disulfide and/or polysulfides are produced from the polysulfurated (persulfurated)
catalytic-site cysteine of MST.
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2. Materials and Methods
2.1. Preparation, overexpression, and purification of rat wild-type MST
Rat recombinant wild-type MST was overexpressed and purified according to a
previously described procedure [10]. Then, MST was mixed with a 10-fold molar
excess of sodium TS (Wako Pure Chemicals, Osaka, Japan) in 20 mM potassium
phosphate buffer, pH 7.4 (sample A) to maintain active form of MST, and was frozen
until use.
2.2. Removal of an outer sulfur/polysulfur (of a persulfide/polysulfide formed at Cys247
of TS-pretreated MST) by cyanide
A 50- l solution containing 40 l of MilliQ water (Merck Millipore, Darmstadt,
Germany) and 10 l of 3.5 mM TS-pretreated MST (sample A) was incubated with 3.5
l of 1 M potassium cyanide (KCN, Wako Pure Chemicals, Osaka, Japan) on ice for
2.5 hr. After the excess of cyanide was removed by gel-filtration on a G-25 column (1 ×
24 cm, GE Healthcare BioSciences, Uppsala, Sweden), the enzyme-containing fractions
were collected and concentrated using a VIVASPIN (molecular weight cutoff [MWCO]:
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10,000; Sartorius, Goettingen, Germany) to 20 l (sample B).
2.3. Addition of a sulfur/polysulfur to form a persulfide/polysulfide at Cys247 of
CN-pretreated MST
A 50- l solution containing 40 l of MilliQ water and 10 l of 3.5 mM
CN-pretreated MST was incubated with 3.5 l of 1 M TS on ice for 2.5 hr. Excess TS
was removed by gel filtration on a PD10 column (GE Healthcare BioSciences, Uppsala,
Sweden), and the enzyme-containing fractions were collected and concentrated with
VIVASPIN (MWCO: 10,000) to 20 l (sample C).
2.4. Determination of chemical forms of Cys247 by Matrix-assisted laser desorption
ionization tandem time-of-flight mass spectrometry
Each sample was diluted to 1 M with MilliQ water (salt concentration had to be
less than 2 mM). An appropriate aliquot (less than 0.5 ml) of each sample in 70%
acetonitrile (Wako Pure Chemicals) containing 0.1% trifluoroacetic acid (Wako Pure
Chemicals) was mixed with 0.5 l of Supper-DHB (Bruker Daltonik GmbH, Bremen,
Germany) 10 mg/ml in 30% acetonitrile containing 0.1% trifluoroacetic acid. The
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mixture was dried at room temperature on the target plate.
Matrix-assisted laser desorption ionization tandem time-of-flight mass
spectrometric (MALDI-TOF/TOF MS) analysis was conducted using an ultrafleXtreme
(Bruker Daltonik GmbH) equipped with the smartbeam-II laser operating in the linear
positive mode with 25-kV acceleration voltage. Mass spectra were obtained by
averaging 20,000 individual laser shots. External mass calibration was performed using
a protein mixture called Protein Calibration Standard II (Bruker Daltonik GmbH)
containing protein A and trypsinogen; (m/z 44,613; [M + H]+) and (m/z 23,482; [M +
H]+), respectively. Peak assignment across the entire mass range and/or on its shoulder
in an extended spectrum is reliable, as discussed previously [10,11]. Peak determination
over the entire mass range and/or on its shoulder in an extended spectrum is reliable as
described previously [10, 12]. The mass measurement error was less than 0.037% for
the mass spectrometric analysis of a chemical modification of 30–40 kDa protein.
2.5. Removal and production of H2S and polysulfides from modified Cys247 by reduction
A 60- l solution containing 50 l of MilliQ water and 10 l of 3.5 mM control
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MST (TS-preincubated MST, sample A) was prepared. TS-treated MST (Sample C) was
prepared according to the experimental procedure described above. The mixture was
supplemented with E. coli wild-type thioredoxin (Trx) or E. coli mutant Trx (C32S:
Cys32 is replaced with Ser) (a gift form Dr. Matsumura, Dep. of Biochemistry, Nippon
Medical School), with a Trx-reducing system containing E. coli Trx reductase (TRD,
Sigma-Aldrich, St. Louis, MO, USA), and NADPH (Sigma-Aldrich) (MST:C32S or
Trx:TRD:NADPH = 35 nM to 175 nM or 175 nM to 0.7 nM to 437.5 nM) [11]. The
reason why E. coli Trx and TRD were used because they are much more effective than
mammalian Trx and TRD [11]. Each mixture was incubated on ice for 90 min. Next, the
Trx-supplemented sample with a Trx reducing system was incubated on ice for 24 hr.
Each reaction was stopped by filtration through Amicon Ultra-0.5 (3 kD, Merck
Millipore, Darmstadt, Germany), i.e., by separation of these reagents from MST. Then,
the filtrates were referred to as samples D, E, and F, respectively.
2.6. Derivatization of each product with monobromobimane
Each sample (A, C, D, E, and F) was adjusted to 75 l with MilliQ water and
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supplemented with 25 l of 0.1 M Tris-HCl buffer (pH 8.0). After that, 4 l of 50 mM
monobromobimane (mBB, Merck Millipore) was added to the solution. Each mixture
was incubated at 25 °C for 30 min in the dark. After addition of 10 l of 25%
sulfosalicylate (Nacalai Tesque, Kyoto, Japan), each mixture was incubated on ice for
15 min in the dark. After that, the samples were frozen until analysis and centrifuged
before analysis.
2.7. Determination of chemical structure of derivatized products by liquid
chromatography-tandem mass spectrometry
Samples derivatized with mBB were analyzed by a triple-quadrupole mass
spectrometer coupled to high-pressure liquid chromatography (HPLC) system
(LCMS-8040, Shimadzu, Kyoto, Japan). The samples were loaded onto a reverse-phase
Symmetry C18 HPLC column (4.6ꢀ×ꢀ250ꢀmm, Waters, Milford, MA, USA) and run at
the flow rate of 0.8ꢀml/min. The mobile phase consisted of (A) 5 mM ammonium
formate (Wako Pure Chemicals) and (B) 5 mM ammonium formate:methanolꢀ=ꢀ1:1. The
samples were separated by eluting with a gradient: 40% B at 0ꢀmin, and 75% B at 8ꢀmin,
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which was then held for 10 min. The column oven was maintained at 40 °C. The
effluent was subjected to the liquid chromatography-tandem mass spectrometry
(LC-MS/MS) using an electrospray ionization (ESI) interface operating in positive-ion
mode. The source temperature was set to 400ꢀ°C, and the ion spray voltage was to
4.5ꢀkV. Nitrogen served as a nebulizer and drying gas. The tandem mass spectrometer
was tuned in the multiple-reaction monitoring mode to monitor mass transitions m/z
Q1/Q3 432.45/192.00 [mBB-S-mBB+NH4]+, 464.55/192.00 [mBB-S2-mBB+NH4]+,
and 496.60/192.00 [mBB-S3-mBB+NH4]+. Analysis of sulfide and polysulfides was
performed by a previously described method using LC-MS/MS method [4] with
modification.
2.8.HPLC-fluorescence analysis
H2S and H2S3 concentrations were measured by a previously described method
using HPLC-fluorescence (FL) with a modification [14, 15]. The chromatographic
system consisted of an L-2130 pump (Hitachi, Tokyo, Japan), an L-7300 column oven
(Hitachi), and an RF-20A fluorescence detector (Shimadzu). Samples were loaded onto
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a reverse-phase Symmetry C18 HPLC column (4.6ꢀ×ꢀ250ꢀmm, Waters) and run at the
flow rate of 0.8ꢀml/min. The mobile phase consisted of (A) 5 mM ammonium formate in
water and (B) 5 mM ammonium formate water:methanolꢀ=ꢀ1:1. The samples were
separated by eluting with a gradient: 40% B at 0ꢀmin, and 75% B at 8ꢀmin, which was
held for 10ꢀmin. The column oven was maintained at 40ꢀ°C. The mBB adduct was
monitored with a scanning fluorescence detector at an excitation wavelength of 370ꢀnm
and emission wavelength of 485ꢀnm.
2.9. The assay of rhodanese activity of MST
Incubation with mercaptopyruvate, 2-mercaptoethanol, and CN may cleave the
disulfide bond in the persulfide and in polysulfides formed at a catalytic-site cysteine.
Therefore, it is not appropriate to measure the catalytic activity using these compounds
as substrates. In this experiment, however, the measurement of the rhodanese activity of
MST [10] was carried out at the first step.
2. 10. Protein quantification
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Protein concentrations were determined using a Coomassie protein assay kit
(Pierce Biotechnology, Inc., Rockford, IL, USA) with crystalline bovine serum albumin
(BSA, MP Biochemicals, Irvine, CA, USA) as the standard.
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3. Results and Discussion
3.1. Persulfide and polysulfides formed at the catalytic-site cysteine
In MALDI-TOF/TOF MS analysis, each profile with extended axis representing
[m/z] [10,12] is shown. In sample A (Fig. 1), MST after incubation with sodium thiosulfate
(TS) yielded three peaks (m/z 32,791.1; [M + H]+) for nonmodified MST, (m/z 32,867.6; [M +
H]+) for disulfurated (S2) MST, and (m/z 32,940.3; [M + H]+) for tetrasulfurated (S4) MST, and a
shoulder (ca. m/z 32,830; [M + H]+) for persulfurated (S) MST. Each measurement error in the
mass number of each sulfur atom (38.9, 37.6, 32.4, and 40.3, respectively) is less than 0.025%.
These findings indicate that prolonged incubation with TS increases amounts of polysulfurated
(S2, 3, and 4) MST, especially the S2 modified form. When rat recombinant MST is
overexpressed in E. coli, approximately 60% of this enzyme’s pool is converted to persulfurated
forms, and MST untreated with TS shows two peaks with the molecular masses m/z 32,803 ([M
+ H]+) for unmodified MST and m/z 32,830 ([M + H]+ for persulfurated MST) [12].
In sample B (Fig. 1), sample A after treatment with potassium cyanide (CN), cyanolysis,
decreased a peak (m/z 32,791.1) and a shoulder (ca. m/z 32,830). As a result, sample B yielded
two peaks (m/z 32,796.7; [M + H]+) for unmodified MST and (m/z 32,874.7; [M + H]+) for
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disulfurated (S2) MST and a shoulder (m/z 32,941.1; [M + H]+) for tetrasulfurated (S4) MST.
Each measurement error in mass number of each 2 sulfur atoms (78.0 and 66.4) was less than
0.037%. In our previous study, cyanolysis resulted in cleavage of the outer sulfur of a persulfide
[12]. It is noteworthy that after long incubation with TS, polysulfurated (S2, , and ) MSTs
3
4
become resistant to cyanolysis.
In sample C (Fig, 1), sample B after incubation with TS showed four peaks (m/z 32,791.1;
[M + H]+ for unmodified MST), (m/z 32,824.6; [M + H]+) for persulfurated (S) MST, (m/z
32,867.6; [M + H]+) for disulfurated (S2) MST, and (m/z 32,897.6; [M + H]+) for trisulfurated
(S3) MST. Of four peaks, a peak of S2 MST was newly appeared. Other three peaks were
basically identical to those of sample A. Each measurement error in mass number of each sulfur
atom (33.44, 43.01, 30.00, and ca. 32.40, respectively) was less than 0.034%. It was concluded
that lengthy incubation with TS yields some types of polysulfuration (S2, , and ) at the
3
4
catalytic-site cysteine and the disulfide modification is dominant in this study. It is worth noting
that polysulfide adducts are relatively resistant to cyanolysis.
These findings concluded that long-term treatment with TS converted MST to
(poly)sulfurated reaction intermediate forms. Moreover, persulfurated (S1), disulfurated (S2),
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trisulfurated (S3), and tetrasulfurated (S4) MSTs were observed which was confirmed by
TS-retreatment after cyanolysis.
3.2. Production of hydrogen sulfide/polysulfides from MST that formed persulfide/polysulfides
LC-MS/MS analysis for monobromobimane (mBB)-derivatized compounds revealed that
sample A (control MST) and sample C (TS-treated MST) yielded no peak (Fig. 2). In sample D
(Fig. 2), after incubation of sample C with E. coli mutant Trx, C32S (Cys32 is replaced with Ser)
and a reducing system (MST:C32S:E. coli Trx reductase (TDR):NADPH = 1:5:0.02:12.5 molar
ratio) [11] for 60 min, there was no peak related to polysulfides (Fig. 2). This result was
supported by the finding that C32S did not form a persulfide and/or polysulfide at Cys32 [11].
In sample E (Fig. 2), after sample C (TS-pretreated MST) was treated with Trx
and a Trx reducing system (MST:Trx:TDR:NADPH = 1:5:0.02:12.5) for 60 min, a new
peak (mBB-S2-mBB as a NH4+ form) appeared. These findings concluded that
(poly)sulfurated catalytic-site cysteine after treatment with TS, can be reduced by Trx
with a Trx reducing system for 60 min to form H2S2.
In sample F (Fig.2), after sample A (TS-pretreated MST) was treated with Trx and
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a Trx reducing system for 24 hr, two new peaks appeared: [mBB-S1-mBB] and
[mBB-S3-mBB] in the NH4+ forms. Another peak [mBB-S2-mBB] in the NH4+ form was
also observed, but the intensity was smaller than that in Sample E. These findings
resulted that (poly)sulfurated catalytic-cysteine after treatment with TS, can be reduced
by Trx with a Trx reducing system for 24 hr to form H2S, H2S2, and H2S3.
It was noteworthy that H2S2 appeared at 60 min and then H2S and H2S3 appeared
later. Tetrasulfide was reported to function as sulfuration of a specific protein [13], but
tetrasulfide was not detected by the present LC-MS/MS analysis although
MALDI-TOF/TOF MS analysis showed tetrasulfurated MST formation.
3.3. Semi-quantitative analysis of mBB-derivatized compounds using HPLC-FL
To conform semi-quantitative determination of each peak in LC-MS/MS analysis,
high-pressure liquid chromatography-fluorescence (HPLC-FL) analysis was performed
(Fig 3). However, a peak corresponding to H2S2 [mBB-S2-mBB] cannot be detected in
this procedure [14, 15]. For control, 10 M and 50 M Na2S were applied to the
HPLC-FL (Fig. 3). When 10 M Na2S was applied, an only peak corresponding to H2S
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[mBB-S-mBB] was detected (Fig 3). Then, when 50 M Na2S was applied, two peaks
corresponding to H2S [mBB-S-mBB] and of H2S3 [mBB-S3-mBB] were detected (Fig
3).
Sample F (Fig 3) after incubation of sample C with Trx and a Trx reducing
system for 24 hr, two peaks corresponding to H2S [mBB-S-mBB] and H2S3
[mBB-S3-mBB] appeared. These findings confirmed that the two peaks at 24 hr in the
LC-MS/MS analysis were those of H2S [mBB-S-mBB] and H2S3 [mBB-S3-mBB].
3.4. The mechanism of hydrogen sulfide/polysulfides formation from MST
The findings in this experiment confirmed that H2Sn is produced from reaction
intermediates from persulfurated and polysulfurated catalytic-cysteine of MST via Trx
reduction (Fig. 4), which is supported by the fact that C32S, which lacks formation of a
disulfide bond with a protein [11], did not produce H2Sn. It is noteworthy that H2S2 is
first produced from reaction intermediate of MST via Trx reduction in this production
system. This is consistent with the fact that the dominant polysulfides are disulfides at
pH>7 [16]. After this reaction, two possible reactions proceed to produce H2S and
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H2S3. It is very likely that H2S and H2S3 are produced from H2S2. Two molecules of
H2S2 can be converted to H2S3 [17]. On the other hand, H2S3 and H2S2 are
thermodynamically unstable and decomposed to H2S [17, 18]. The other possibility is
much less likely than the former that H2S and H2S3 are independently produced form
each reaction intermediate of MST, because all experiments but derivatization with
mBB were performed around neutral pH. However, the mechnism of production of
polysulfides is still under study.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgments
This study was supported by JSPS KAKENHI Grant Number JP15K08322.
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Figure legends
Figure 1. MALDI-TOF-MS/MS analysis of the sulfurated catalytic-site cysteine of MST
In sample A (TS-incubated MST), a peak○1 (m/z 32,791.1; [M + H]+) represents
nonmodified MST, a peak○3 (m/z 32,867.6; [M + H]+) dose disulfurated (S2) MST, a
peak○5 (m/z 32,940.3; [M + H]+) does tetrasulfurated (S4) MST, and a shoulder○2 (ca.
m/z 32,830; [M + H]+) does persulfurated (S) MST. In sample B (CN treatment of
TS-incubated MST), a peak○1 (m/z 32,796.7; [M + H]+) represents unmodified MST
and a peak○3 (m/z 32,874.7; [M + H]+) does disulfurated (S2) MST and a shoulder○5
(m/z 32,941.1; [M + H]+) does tetrasulfurated (S4) MST. In sample C (TS treatment of
CN-treated MST), a peak○1 (m/z 32,791.1; [M + H]+) represents nonmodified MST, a
peak○2 (m/z 32,824.6; [M + H]+) does persulfurated (S) MST, a peak○3 (m/z 32,867.6;
[M + H]+) does disulfurated (S2) MST, and a peak○4 (m/z 32,897.6; [M + H]+) does
trisulfurated (S3) MST. Arrows indicate profiles with an extended axis (m/z). Each
measurement error in the mass number of each molecule is less than 0.037%.
Figure 2. LC-MS/MS analysis of mBB-derivatized compounds
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Sample A, TS-incubated MST (control material); Sample C, TS treatment after
CN treatment of MST (Sample B); Sample D, after treatment of TS-treated MST
(Sample C) with C32S, TRD, and NADPH for 60 min; Sample E, after treatment of
TS-treated MST (Sample C) with Trx, TRD, and NADPH for 60 min; Sample F, after
treatment of TS-treated MST (Sample C) with Trx, TRD, and NADPH for 24 hr. The
uppermost column, measurement of [bimane-S-bimane + NH4]+ dissociating into a
precursor ion 432.45 (m/z) and product ion 192 (m/z); the middle column, measurement
of [bimane-S2-bimane + NH4]+ dissociating into the precursor ion 464.55 (m/z) and
product ion 192 (m/z); the bottom column, analysis of [bimane-S3-bimane + NH4]+
dissociating into the precursor ion 496.60 (m/z) and product ion 192 (m/z). Each
molecule was determined according to the method [14, 15].
Figure 3. HPLC-FL analysis of mBB-derivatized compounds
Na2S, a control reagent forming a bimane-S-bimane adduct; Sample E, after
treatment of TS-incubated MST (Sample C) with Trx, TRD, and NADPH for 60 min;
Sample F, after treatment of TS-incubated MST (Sample C) with Trx, TRD, and
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NADPH for 24 hr; S indicates bimane-S-bimane (H2S adduct); S3 denotes
bimane-S3-bimane (H2S3 adduct). The molecule bimane-S2-bimane (H2S2 adduct)
cannot be detected by HPLC-FL analysis. Each molecule was determined according to
the method [14, 15].
Figure 4. The proposed mechanism of formation of H2Sn from reaction intermediates of
MST.
Details are in the text.
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